The present invention relates to a method for the modification of protein structure in finish shaped feed pellets, balls or the like, among other reasons in order to contribute to strengthening the permanence of the pellet shape in granular feeds of this sort. The invention also relates to feedstuff manufactured according to this method for the formation of a shape-permanent feed in pellet form.
Many types of animals in breeding, e.g. salmon and trout, are carnivorous (meat-eating). Their natural food consists of insects (for salmon and trout in the freshwater phase), fish and crustaceans. For other carnivorous farmed animals, such as mink and foxes, the feed may also contain the mammalian flesh, for example fresh slaughterhouse waste. Insects and crustaceans have an exoskeleton which i.e. consists chitin. Chitin is a linear polysaccharide of N-acetyl-D-glucosamine linked by xcex21xe2x86x924 bonds. Other structural carbohydrates such as cellulose (xcex2[1xe2x86x924] D-glycose) and alginate (D-mannuronic acid) are not found in these prey animals, nor do they contain starch (xcex1 [1xe2x86x924] D-glycose) as energy stores. Nevertheless, salmon, trout and other carnivorous fish and animals have enzymes (for example, amylase) that are capable of breaking down starch in the gut and making it digestible, but they may be less efficient in this respect than herbivorous (plant-eating) fish and animals.
In feeds intended for carnivorous fish it is usual to add between 8% and 25% carbohydrates, for example in the form of wheat or maize, as a binding agent. After pressing, but especially after extrusion, the starch in these carbohydrates will form a matrix or base mass which gives the pellets mechanical strength and shape permanents so that the shape of the pellets can be maintained after drying, further processing, storage and transport.
Carbohydrates are utilised in metabolism as a source of energy. The energy density of carbohydrates is lower than that of protein and fat (17.6; 23.9 and 39.8 MJ/kg respectively). The digestibility of carbohydrates is also lower in carnivorous fish, and declines as the proportion of complex carbohydrates in the feed (above 10%) increases. Experiments have shown that salmonids have no metabolic need for carbohydrates. If fat replaces carbohydrates as an energy source, a carbohydrate-free fish feed of this sort will contain more energy per unit weight, as long as the relative proportions of the other components are held constant.
In order to give feed pellets shape permanence and mechanical strength, as mentioned above, it is known to add a binding agent in the form of 8%-25% carbohydrates, for example wheat and/or maize. After pressing or extrusion in the feedstuff material there will be established a starch matrix of the desired strength.
Other techniques in connection with the forming of feed into pellets balls or the like, have also been described. According to U.S. Pat. No. 4,935,250, for example, a gel or mass of alginate is also produced during the forming.
The patent literature includes descriptions of feeds and feed mixtures in which the mass consists of gelatine or caseinate. See, for example, British Patent No. 2,217,175.
There are also feeds in which the binding characteristics produced by the coagulation of native proteins are exploited; see NO 179 731.
Small feed particles can be produced with the aid of an agglomeration technique, which are based on the principle of aggregating extremely small particles into larger particles. This process does not utilize carbohydrates as a binding agent. The feed components are bound together through various forms of contact bonds between the solid particles in the feed. The different forms of contact bonds can vary from hydrogen bonds, adhesion and cohesion to capillary forces. New covalent bonds are not created in this process. This is an obvious disadvantage for the maintenance of the feed pellet""s form and strength, because covalent bonds are stronger than other chemical bonds.
A serious disadvantage of agglomerated feeds is thus that the bonds are weak, and given the lack of a continuous matrix such pellets are friable and fragile. The agglomeration technique cannot be utilised to produce particles of feed in pellet form with a diameter larger than about 2.5-3.0 mm.
In order to be able to produce larger feed particles/fragments/pellets/balls, etc., we must abandon the agglomeration technique without addition of carbohydrate and again return to carbohydrate as a binding agent. In low concentrations, complex carbohydrates such as strarch are digested by salmonids, for example, but if their concentration exceeds 10% the digestibility of the carbohydrate fraction decreases (Aksnes A., 1995. Growth, feed efficiency and slaughter quality of salmon, Salmo salar L., given feed with different ratios of carbohydrate and protein. Aquaculture Nutrition, 1:241-248).
The energy content of the carbohydrate fraction may be replaced by fat with the feed recipe. This will result in greater freedom with respect to varying the relative proportions of fat, protein and micronutrients since the carbohydrates make up the reminder of the feed recipe. Such a fed will be richer in energy than an equivalent feed containing carbohydrates, and a reduction in the feed conversion ratio, defined as the quantity of feed consumed to produce one kilo of fish biomass, will be obtained.
In accordance with the present invention one has aimed at showing a method of modifying the protein structure of feeds whose nutrient composition closely resembles the natural choice of foods of carnivorous fish and animals. According to the invention, favourable binding is obtained in feedsxe2x80x94without carbohydratesxe2x80x94resulting in shape permanence in pellets and similar forms of feedstuff, and in such a way that the maximum particle size/pellet diameter can be increased in the case of agglomerated feed while maintaining the shape of the feed. The invention also aims to increase the energy density of all types of feed, particularly fish feeds.
The above identified objective is reached by proceeding as described herein. A feedstuff that has been treated as described in accordance with this method and which is intended for the forming of pellets or the like, and properties relating thereto are identified herein. Feed pellets made of such a feedstuff, whose protein structure has been modified by the method, are also disclosed herein.
The method and product formed by the method thus consists mainly of a feedstuff, or alternatively of one or more of the components of which it consists, for forming pellets, alternatively of coating the surface of the formed pellets, in order to add an enzyme (preferably transglutaminase) that function as a catalyst for catalyses the formation of covalent bonds between the amino acids which make up the protein chains in protein raw materials containing proteins in native or denatured form. The raw materials of the protein in the feed mixture include the natural amino acids glutamine and lysine in the protein chains. The added enzyme will act as a catalyst and catalyze the formation of covalent (xcex5-(xcex3-Glu)Lys) bonds between the amino acids glutamine and lysine in the protein raw ingredients in the feed.
Through the adoption of reaction temperature and reaction time this enzymatic reaction will form a matrix or basic mass of protein raw materials which will exhibit adequate strength to give feed pellets a constant and lasting shape.
In agglomerated feed this enzymatic reaction will lead to covalent transverse bonds (cross-bonds) between the proteins. This will give the agglomerate increased strength by introducing the strongest type of chemical bond in addition to the other three types of chemical bonds which provide the feed product with firmness and strength.
In pressed or extruded feeds, the formation of covalent (xcex5-(xcex3-Glu)Lys) bonds that lead to the formation of a protein matrix will be able to partially or wholly replace the addition of carbohydrates. This makes it possible to remove carbohydrates from the recipe for the feed, or to reduce the proportion of carbohydrates respectively. Carbohydrates play virtually no part in the natural diet of carnivorous fish and animals, and the total energy density of feed pellets can be increased because both protein and fat are richer in energy than carbohydrates. According to present invention, an animal feed in the form of pellets will require no other binding agents such as gelling agents in the form of gelatine, for example. 
The above reaction formula, in which the enzyme transglutaminase acts as a catalyst, shows the creation of xcex5-(xcex3-Glutamyl) lysine bond between the amino acids glutamine and lysine.
Transglutaminase is defined as enzymes which are classified as protein-glutamine xcex3-glutamyltransferase (EC 2.3.2.13; International Union of Biochemistry and Molecular Biology, Nomenclature Committee). Transglutaminase may occur in a pure form or as a distinct premix with suitable filler and transglutaminase in adjusted concentration. Transglutaminase can be added to the other raw ingredients of the feed in the form of a powder, in solution or in suspension.
The term xe2x80x9cprotein raw materialsxe2x80x9d refers to raw ingredients that contain protein in either native or denatured form. Examples include fish meal, stickwater, stickwater concentrate, blood meal, feather meal, bone and horn meal, wheat gluten, maize gluten, soya meal and rapeseed meal. These are only illustrative examples, and do not exclude the use of other raw ingredients in feeds as partial substitutes for or additions to one or more of the said raw ingredients.
The term xe2x80x9cfeed pelletsxe2x80x9d refers to particles or fragments, preferably round, which are formed by means of a special process, such that they are of a size and shape that makes them suitable as feed, particularly for carnivorous fish species such as farmed salmon, cod, halibut, sea perch and sea bream.
The activity of the enzyme (transglutaminase) declines at temperatures higher than 50xc2x0 C., and it is deactivated at temperatures beyond 65xc2x0 C. The formation of a protein matrix as described here can thus take place under process heat conditions in which the process temperature varies between 0 and 60xc2x0 C. The shortest process time is obtained at around 50xc2x0 C., since the process time is lengthened at temperatures above and below this temperature.
Transglutaminase is added to the other ingredients of the feed before these are formed into pellets by a suitable method, since the enzyme can be added as a solution or as a suspension in a suitable liquid, for example water, or blended as a dry ingredient before liquid is added to the mixture. In addition to transglutaminase, an aqueous solution may contain pure water, stickwater or stickwater concentrate, or another protein-rich liquid, for example a non-limiting gelatine solution.
Transglutaminase can also be added to the surface of preformed pellets by a suitable method, in that the enzyme can be added as dissolved in steam, or as a solution, suspension or wash in a suitable liquid such as water.
The fat content of the feed may, according to a non-limiting preferred example, consist of fish-oil, which can be added to the feed either before or after the feed it is formed into pellets, or after the pellets these have been dried. After the feed has been formed into pellets, the pellets are maintained at a temperature of between 0 and 60xc2x0 C., so that the transglutaminase enzyme has time to catalyse the xcex5-(xcex3-Glutamyl) lysine bonds that are desired.
The reaction time is adapted to the reaction temperature.
Finally, the formed pellets are dried to the desired water content/degree of dryness in a suitable dehydration unit, such as a drying cabinet.
The transglutaminase product used in the following examples (from the manufacturer Ajinomoto in Japan) consists of 60% sodium caseinate, 39.5% maltodextrin and 0.5% transglutaminase. A meat grinder with a die size of around 6.5 mm can be used to form the feed into pellets. The apparatus may also comprise an incubation cabinet and a drying cabinet (working temperature about 80-90xc2x0 C.). In a wear test, a rotation rate of 500 rpm was employed without the use of metal balls.